The present invention relates to somatotropin analogues with amino acid changes in the alpha-helix 1, alone or in combination with mutations with the alpha-helix 3 and/or alpha-helix 2 portion of said somatotropins and to methods for producing the changes in the alpha-helix 1, as well as other regions, of recombinantly-produced polypeptides or proteins. Administration of exogenous somatotropins significantly increases the growth performance of a variety of animals, in particular livestock animals such as swine, but also fish species, as well. This growth enhancement in livestock in particular is usually characterized by an increase in muscle mass accretion concomitant with a decrease in fat, resulting in larger, leaner animals. The feed efficiency of animals receiving exogenous somatotropin also is significantly improved, resulting from an increase in weight gain and a concomitant decrease in feed consumption.
Exogenous administration of somatotropin is achieved in several ways, such as daily injections. In certain instances, however, other routes of administration may be preferred. For instance, an implanted device which allows sustained release of somatotropin over a defined time period may be helpful when treating certain livestock. A more desired route of administration is via an implanted device that allows sustained release over a defined period of time. Such a device would contain large amounts of somatotropin in very high concentrations (ca 500 mg/ml). Further, a somatotropin molecule having high solubility and a low tendency to form insoluble, biologically inactive aggregates is required for such delivery uses.
Somatotropins contain four .alpha.-helices which assemble to form an .alpha.-helical bundle (Abdel-Meguid et al, 1987). Typically, amino acid side chains projecting into the core of this structure are non-polar, hydrophobic and very tightly packed together in order to exclude penetration of a polar solvent (such as water or saline) into the center of the bundle. In the case of bovine somatotropin, which is related to porcine somatotropin in primary sequence, exposure of the hydrophobic face of .alpha.-helix 3 (from amino acid residues tyr.sub.110 to leu.sub.127) under protein concentrations in excess of 1 mg/ml promotes the formation of "associative intermediates", which are hypothesized to be a nucleating event in aggregate formation (Brems et al, 1986; Brems, 1988). These associative intermediates may represent alternate packing arrangements of this .alpha.-helix from several individual somatotropin molecules, resulting in a multimeric structure in which the hydrophobic faces of this helix are resequestered from the aqueous environment. Formation of the associative intermediates can be blocked by addition of an excess of a protein fragment-containing .alpha.-helix 3 (Brems, et al, 1986). In addition, extending the hydrophobic face of this helix, by replacing lysine at position 112 with leucine, greatly exacerbates the tendency to form associative intermediates (Brems, 1988).
The present invention addresses the problem of low solubility of somatotropins by altering the .alpha.-helix 3 regions of the somatotropins. Specifically, porcine somatotropins with enhanced solution stability in vitro are made by site-directed mutagenesis of .alpha.-helix 3. Both the hydrophobic and hydrophilic faces are targeted for mutagenesis. Recently site-directed mutations in the .alpha.-helix 3 region of bovine somatotropin resulted in suppressed growth of transgenic mice expressing the mutant somatotropin, a result suggesting that the .alpha.-helix 3 region is a region important for which biological activity may be maintained (Chen et al, 1990).
In addition, .alpha.-helix 3 mutations are combined, where appropriate, with mutations in the helix 1 or helix 2 regions, and with double mutations in the DNA encoding cysteine at positions 183 and 191, where DNA encoding cysteine is replaced with either alanine or glutamic acid encoding DNA. The double mutations at positions 183 and 191 are described in EP355460. Through the use of the mutations disclosed herein, somatotropins with enhanced solubility (stability), and thereby enhanced properties for sustained release, are provided. Porcine somatotropin is particularly useful in a sustained release form, and as such is a somatotropin of primary interest.
A particularly useful example of the present mutation is mutation I122L, in which the isoleucine at position 122 in .alpha.-helix 3 is replaced with leucine. In combination with other mutations at positions 183 and 191 where the cysteines are replaced by alanine, a significant increase in the transition temperature of the protein's single tryptophan residue is obtained. The transition temperature is a measure of the thermal stability of the protein. In one of the most preferred mutation, enhanced solution stability is obtained when the I122L mutation is combined with mutations in which the cysteine-encoding DNA at positions 183 and 191 are altered to encode glutamic acid. In another preferred mutation, enhanced solution stability is achieved when the helix I double mutant A6TS11R is combined with mutations in which the cysteine-encoding DNA at positions 183 and 191 in the amino acid sequence are altered to encode glutamic acid.